Effects of long term denervation on smooth muscle of the chicken expansor secundariorum

Summary Denervation of the expansor secundariorum muscle of the adult and 2 week chicken, by sectioning the brachial plexus, resulted in an approximate twofold increase in dry weight over 8 weeks. Unlike skeletal muscle, no ultrastructural changes were exhibited by the smooth muscle cells for a period of up to 5 months post denervation. No evidence of hypertrophy of the individual muscle cells was observed, but following colchicine treatment a definite increase in the number of mitotic figures was noted within muscle bundles indicating that the increase in dry weight of the expansor muscle is due to hyperplasia of the smooth muscle cells. The results are discussed in relation to in vitro studies of the interaction of sympathetic nerves with smooth muscle.     

Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta

The potency, structure-activity relationship, and mechanism of vasorelaxation of a series of flavonoids, representing different subclasses (flavonols: fisetin, rutin, quercetin; flavones: chrysin, flavone, baicalein; flavanones: naringenin, naringin; isoflavones: diadzein and flavanes: epigallo catechin gallate), were examined in the isolated rat aorta. Most of the flavonoids tested showed concentration dependent relaxant effects against K+ (80 mM) and phenylephrine (PE, 0.1 microM)-induced contractions with a greater inhibition of the responses to the alpha1-adrenoceptor agonist. The relaxant effects of most of the flavonoids involve in part the release of nitric oxide and prostaglandins from the endothelium as pretreatment with L-NAME and indomethacin attenuated the responses. In addition, the relaxant action of the flavonoids includes inhibition of Ca+2 influx and release of Ca+2 from intracellular stores. A structure-activity relationship amongst the flavonoids was suggested.     

Effects of nicorandil on isolated smooth muscle cells from guinea-pig taenia caeci

1. The effects of nicorandil on guinea-pig taenia caeci were investigated with the use of isolated smooth muscle cells and glycerin-treated muscle fiber bundles. 2. Nicorandil inhibited high K-, Ca2+- and carbachol-induced contractions in a dose-dependent manner without affecting 45Ca fluxes in isolated cells. 3. Nicorandil had no effect on ATP-induced contraction of glycerin-treated muscle fiber bundles. 4. The present results suggest that nicorandil may inhibit the contraction by action on the contractile proteins in an indirect manner in guinea-pig taenia caeci.     

Effects of load and tone on the mechanics of isolated human bronchial smooth muscle

Isotonic and isometric properties of nine human bronchial smoothmuscles were studied under various loading and tone conditions. Freshlydissected bronchial strips were electrically stimulated successively atbaseline, after precontraction with10⁷ M methacholine (MCh),and after relaxation with10⁵ M albuterol (Alb).Resting tension, i.e., preload determining optimal initial length(L_o) atbaseline, was held constant. Compared with baseline, MCh decreasedmuscle length to 93 ± 1%L_o(P < 0.001) before any electricalstimulation, whereas Alb increased it to 111 ± 3%L_o(P < 0.01). MCh significantlydecreased maximum unloaded shortening velocity (0.045 ± 0.007 vs.0.059 ± 0.007 L_o/s), maximalextent of muscle shortening (8.4 ± 1.2 vs. 13.9 ± 2.4%L_o), and peakisometric tension (6.1 ± 0.8 vs. 7.2 ± 1.0 mN/mm²). Alb restored all thesecontractile indexes to baseline values. These findings suggest that MChreversibly increased the number of active actomyosin cross bridgesunder resting conditions, limiting further muscle shortening and activetension development. After the electrically induced contraction,muscles showed a transient phase of decrease in tension below preload.This decrease in tension was unaffected by afterload levels but wassignificantly increased by MCh and reduced by Alb. These findingssuggest that the cross bridges activated before, but not during, theelectrically elicited contraction may modulate the phase of decrease intension below preload, reflecting the active part of resting tension.     

Double-immunofluorescent staining of isolated smooth muscle cells

Summary Contractile proteins have been co-localized by double-immunofluorescent staining in several types of cultured cells. Since freshly isolated smooth muscle cells are more representative of the organization within smooth muscle cells in the intact tissue than cultured cells, the present study was undertaken to determine the feasibility of using double-staining techniques in freshly isolated cells. A new method of purifying -actinin from chicken gizzards was used to provide antigen for raising anti--actinin. Fluorescein isothiocyanate-labelled anti--actinin (FAA) was used in conjunction with tetramethyl rhodamine isothiocyanate-labelled anti-myosin (TRAM) Ouchterlony gels against myosin, tropomyosin, actin, and -actinin showed that antimyosin reacted only with myosin, anti--actinin only with -actinin. Anti--actinin stained only the Z-line of isolated chicken skeletal muscle myofibrils. FAA stained bright, discrete patches or strips on the plasma membrane, while TRAM was excluded from these areas. FAA stained myofibrils faintly in a striated pattern, while TRAM stained myofibrils heavily with less evident striations. Evidence for extramyofibrillar localization of -actinin within the cytoplasm was inconclusive. Although antibodies were quite specific in their labelling, resolution with double-staining was subject to the same limitations described for single labelling of whole cells (Bagby and Pepe 1978). Double-staining of whole cells is just as feasible as single-staining. Indeed, having a definite marker for myofibrils (TRAM) makes the localization of -actinin much easier to interpret.     

纳豆枯草杆菌培养滤液对家兔离体肠平滑肌的作用

目的观察纳豆枯草杆菌培养滤液（culture filtrate,CF）对家兔离体肠平滑肌的作用,并初步探讨其作用机制。方法制备兔离体回肠标本,分别记录肠平滑肌的正常收缩张力和收缩频率作为给药前对照,然后按累积剂量分别加入CF小剂量（每次0．2mL）、CF大剂量（每次0．5mL）、肉汤（每次0．5mL）,给药间隔3min,共给药8次,并描记收缩曲线。观察不同剂量cF对肠平滑肌的作用。另取肠段按毛果芸香碱、CF或阿托品、再毛果芸香碱的顺序给药,观察CF对M胆碱受体的作用。结果CF小剂量组在累积给药达1．6mL时,CF大剂量组在累积给药达3．5mL和4．0mL时,兔离体肠平滑肌收缩张力下降,与给药前比较差异有统计学意义（P〈0．05）,其余各点差异均无统计学意义（P〉0．05）。CF小剂量组在累积给药达1．2、1．4、1．6mL时,CF大剂量组在累积给药达4．0mL时,兔离体肠平滑肌收缩频率明显降低,与给药前比较差异有统计学意义（P〈0．05或P〈0．01）,其余各点差异均无统计学意义（P〉0．05）。CF或阿托品可明显对抗毛果芸香碱引起的兔离体肠平滑肌收缩张力的增加（P〈0．05或P〈0．01）,同时CF还能使其收缩频率明显减少（P〈0．01）。结论纳豆枯草杆菌CF能明显抑制兔离体肠平滑肌蠕动,其作用机制可能与阻断M胆碱受体有关。     

Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

A growing body of data supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues. The increase in the proportion of filamentous actin that occurs in response to the stimulation of smooth muscle cells and the essential role of stimulus-induced actin polymerization and cytoskeletal dynamics in the generation of mechanical tension has been convincingly documented in many smooth muscle tissues and cells using a wide variety of experimental approaches. Most of the evidence suggests that the functional role of actin polymerization during contraction is distinct and separately regulated from the actomyosin cross-bridge cycling process. The molecular basis for the regulation of actin polymerization and its physiological roles may vary in diverse types of smooth muscle cells and tissues. However, current evidence supports a model for smooth muscle contraction in which contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins at the membrane, and proteins within this complex orchestrate the polymerization and organization of a submembranous network of actin filaments. This cytoskeletal network may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. Better understanding of the physiological function of these dynamic cytoskeletal processes in smooth muscle may provide important insights into the physiological regulation of smooth muscle tissues.     

Pharmacology of peptide leukotrienes on ferret isolated airway smooth muscle

The contractile activities of peptide leukotrienes (LT) on isolated spiral strips of ferret trachea were characterized pharmacologically. LTC4 and LTD4 contracted ferret tracheal strips in a concentration-related manner and were 3- to 8-fold more potent than carbachol. In contrast, high concentrations of LTE4 evoked either weak contractions or none at all, whereas LTC4 and D4 were partial agonists compared to carbachol. In tissues which were unresponsive to LTE4, this compound antagonized contractile responses to LTC4 and D4 in an apparently competitive manner: Carbachol-induced contractions were not altered by LTE4. The cyclooxygenase inhibitor, indomethacin (5 microM), LT antagonist, FPL55712 (10 microM), atropine (1 microM), phenoxybenzamine (10 microM), and LTB4 (10 microM) failed to alter LTC4 and D4 concentration-response curves. The results indicate that ferret trachea is sensitive to the contractile activity of LTC4 and LTD4 but not LTE4. The LT-induced contractions appear to be mediated by a direct action of the LT rather than indirectly through release of secondary mediators such as thromboxane, prostaglandin, or acetylcholine. LT receptors in ferret trachea are insensitive to FPL55712 but are antagonized by LTE4.     

Pharmacology of peptide leukotrienes on ferret isolated airway smooth muscle

The contractile activities of peptide leukotrienes (LT) on isolated spiral strips of ferret trachea were chracterized pharmacologically. LTC₄ and LTD₄ contracted ferret tracheal strips in a concentration-related manner and were 3- to 8-fold more potent than carbachol. In contrast, high concentrations of LTE₄ evoked either weak contraction or none at all, whereas LTC₄ and D₄ were partial agonists compared to carbachol. In tissues which were unresponsive to LTE₄, this compound antagonized contractile responses to LTC₄ and D₄ in an apparently competitive manner: Carbachol-induced contractions were not altered by LTE₄. The cyclooxygenase inhibitor, indomethacin (5 μM), LT antagonists, FPL55712 (10 μM), atropine (1 μM), phenoxybenzamine (10 μM), and LTB₄ (10 μM) failed to alter LTC₄ and D₄ concentration-response curves. The results in dicate that ferret trachea is sensitive to the contractile activity of LTC₄ and LTD₄ but not LTE₄. The LT-induced contractions appear to be mediated by a direct action of the LT rather than indirectly through release of secondary mediators such as thromboxane, prostaglandin, or acetylcholine. LT receptors in ferret trachea are insensitive to FPL55712 but are antagonized by LTE₄.     

Effects of membrane potential on just detectable movement in rat skeletal muscle: Effects of denervation

The potential, Vt, at which a brief test depolarization first elicited movement was determined using two-microelectrode point voltage clamp. We expected that inactivation of excitation-contraction coupling at conditioning potentials between ?60 and 0 mV would shift Vt to more positive potentials, and that fibers would become inactivatable with less conditioning depolarization in EDL than soleus. The curve relating Vt to conditioning potential had a negative slope (which was insensitive to addition of 1 mm cobalt or replacement of calcium with 20 mm CaEGTA) between ?60 and ?35 mV and a steep positive slope with further depolarization. Unexpectedly, fibers became inactivatable with less conditioning depolarization in soleus than in EDL when Vt was measured with 50 msec test pulses. However, the positive shift in Vt became less steep as test pulse duration lengthened in soleus fibers. When Vt obtained with test pulses approaching rheobase (10 msec in EDL and 500 msec in soleus) was compared, EDL fibers became inactive with less conditioning depolarization than soleus fibers. The increase in Vt became steeper with 1 mm cobalt or 20 mm CaEGTA and was shifted to more positive potentials by denervation in soleus fibers. We conclude that inactivation (i) does not strongly influence threshold contractions at conditioning potentials between ?60 and ?40 mV and (ii) influences Vt between ?40 and 0 mV in a manner that depends on test pulse duration.     

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

Summary It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3--hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.